[Clinical utility of Genome Boards for patients with complex genetic diseases]. / Utilité clinique des genome boards pour les maladies génétiques complexes.

Genetic analysis using high-throughput sequencing is a powerful tool for patients with rare diseases. However, biological and clinical interpretation thereof is difficult, especially when the clinical picture is complex. Multidisciplinary Genome Boards bring together the relevant medical specialties around the patient's medical and genetic file, to optimize the correlation between phenotype and genotype. This often allows the identification of the causal genetic variant in previously unsolved cases. A retrospective study shows that Genome Boards significantly increase the diagnostic rate in complex clinical cases with difficult-to-interpret genetic analysis results, as well as facilitating collaboration between the various medical specialties involved.

Les analyses génétiques par séquençage à haut débit sont un outil puissant pour les patients atteints de maladies rares. Leurs interprétations biologique et clinique sont cependant difficiles, et cela d'autant plus que le tableau clinique est complexe. Les genome boards multidisciplinaires réunissent les spécialités médicales pertinentes autour du dossier médical et génétique du patient, afin d'optimiser la corrélation entre le phénotype et le génotype. Ceci permet souvent d'identifier le variant génétique causal dans des cas jusque-là non élucidés. Une étude rétrospective montre que les genome boards permettent d'augmenter le taux de diagnostic moléculaire pour des cas cliniques complexes avec des résultats d'analyses difficiles à interpréter, en plus de faciliter la collaboration entre les différentes spécialités médicales impliquées.

Benefits of Exome Sequencing in Children with Suspected Isolated Hearing Loss.

PURPOSE: Hearing loss is characterized by an extensive genetic heterogeneity and remains a common disorder in children. Molecular diagnosis is of particular benefit in children, and permits the early identification of clinically-unrecognized hearing loss syndromes, which permits effective clinical management and follow-up, including genetic counselling. METHODS: We performed whole-exome sequencing with the analysis of a panel of 189 genes associated with hearing loss in a prospective cohort of 61 children and 9 adults presenting mainly with isolated hearing loss. RESULTS: The overall diagnostic rate using exome sequencing was 47.2% (52.5% in children; 22% in adults). In children with confirmed molecular results, 17/32 (53.2%) showed autosomal recessive inheritance patterns, 14/32 (43.75%) showed an autosomal dominant condition, and one case had X-linked hearing loss. In adults, the two patients showed an autosomal dominant inheritance pattern. Among the 32 children, 17 (53.1%) had nonsyndromic hearing loss and 15 (46.7%) had syndromic hearing loss. One adult was diagnosed with syndromic hearing loss and one with nonsyndromic hearing loss. The most common causative genes were STRC (5 cases), GJB2 (3 cases), COL11A1 (3 cases), and ACTG1 (3 cases). CONCLUSIONS: Exome sequencing has a high diagnostic yield in children with hearing loss and can reveal a syndromic hearing loss form before other organs/systems become involved, allowing the surveillance of unrecognized present and/or future complications associated with these syndromes.

Bi-allelic loss of ERGIC1 causes relatively mild arthrogryposis.

Arthrogryposis describes the presence of multiple joint-contractures. Clinical severity of this phenotype is variable, and more than 400 causative genes have been proposed. Among these, ERGIC1 is a recently reported candidate encoding a putative transmembrane protein of the ER-Golgi interface. Two homozygous missense variants have been reported in patients with relatively mild non-syndromic arthrogryposis. In a consanguineous family with two affected siblings presenting congenital arthrogryposis and some facial dysmorphism we performed prenatal array-CGH, postnatal targeted exome and genome sequencing. Genome sequencing identified a homozygous 22.6 Kb deletion encompassing the promoter and first exon of ERGIC1. mRNA quantification showed the complete absence of ERGIC1 expression in the two affected siblings and a decrease in heterozygous parents. Our observations validate the pathogenic role of ERGIC1 in congenital arthrogryposis and demonstrate that complete loss of function causes a relatively mild phenotype. These findings will contribute to improve genetic counseling of ERGIC1 mutations.

Signaling pathways implicated in the stimulation of beta-cell proliferation by extracellular matrix.

Laminin-5-rich extracellular matrix derived from 804G cells (804G-ECM) induces spreading, improves glucose-stimulated insulin secretion, and increases survival and proliferation of rat pancreatic beta-cells. The aim of the study was to determine growth signaling pathways activated by ECM with a particular focus on Ca(2+)-dependent transcription factors. 804G-ECM increased rat beta-cell proliferation, and this stimulation was glucose and Ca(2+) dependent. NF-kappaB nuclear translocation as well as IkappaBalpha gene expression were also Ca(2+) dependent. Inhibition of NF-kappaB almost completely blocked 804G-ECM-stimulated beta-cell proliferation as did the soluble IL-1 receptor antagonist IL-1Ra. 804G-ECM-induced proliferation was also blocked by cyclosporin A and the VIVIT peptide, suggesting involvement of nuclear factor of activated T cells (NFAT)/calcineurin. Use of selective inhibitors further implicated other pathways in this process. Inhibition of phosphatidylinositol 3-kinase and protein kinase A both prevented beta-cell replication stimulated by 804G-ECM. Conversely, inhibition of MAPK, c-Jun N-terminal kinase, p38, and glycogen synthase kinase-3beta increased beta-cell proliferation on 804G-ECM. Our results suggest that Ca(2+) entry, which is necessary for increased beta-cell proliferation on 804G-ECM, is also involved in 804G-ECM-induced NF-kappaB activity. It is proposed that increased cytosolic Ca(2+) leads to activation of the transcription factors NFAT and NF-kappaB that in turn increase beta-cell proliferation. Activation of phosphatidylinositol 3-kinase by 804G-ECM also increases proliferation possibly by synergistic coactivation of NFAT via inhibition of glycogen synthase kinase-3beta, whereas IL-1beta may amplify the process by feed-forward activation of NF-kappaB. Conversely, inhibition of the MAPK pathway increased beta-cell proliferation, indicating a counterregulatory restraining role for this signaling pathway.

Role of the Rho-ROCK (Rho-associated kinase) signaling pathway in the regulation of pancreatic beta-cell function.

Extracellular matrix has a beneficial impact on beta-cell spreading and function, but the underlying signaling pathways have yet to be fully elucidated. In other cell types, Rho, a well-characterized member of the family of Rho GTPases, and its effector Rho-associated kinase (ROCK), play an important role as downstream mediators of outside in signaling from extracellular matrix. Therefore, a possible role of the Rho-ROCK pathway in beta-cell spreading, actin cytoskeleton dynamics, and function was investigated. Rho was inhibited using a new cell-permeable version of C3 transferase, whereas the activity of ROCK was repressed using the specific ROCK inhibitors H-1152 and Y-27632. Inhibition of Rho and of ROCK increased spreading and improved both short-term and prolonged glucose-stimulated insulin secretion but had no impact on basal secretion. Inhibition of this pathway led to a depolymerization of the actin cytoskeleton. Furthermore, the impact of the inhibition of ROCK on stimulated insulin secretion was acute and reversible, suggesting that rapid signaling such as phosphorylation is involved. Finally, quantification of the activity of RhoA indicated that the extracellular matrix represses RhoA activity. Overall these results show for the first time that the Rho-ROCK signaling pathway contributes to the stabilization of the actin cytoskeleton and inhibits glucose-stimulated insulin secretion in primary pancreatic beta-cells. Furthermore, they indicate that inhibition of this pathway might be one of the mechanisms by which the extracellular matrix exerts its beneficial effects on pancreatic beta-cell function.

Interleukin-6 regulates pancreatic alpha-cell mass expansion.

Interleukin-6 (IL-6) is systemically elevated in obesity and is a predictive factor to develop type 2 diabetes. Pancreatic islet pathology in type 2 diabetes is characterized by reduced beta-cell function and mass, an increased proportion of alpha-cells relative to beta-cells, and alpha-cell dysfunction. Here we show that the alpha cell is a primary target of IL-6 actions. Beginning with investigating the tissue-specific expression pattern of the IL-6 receptor (IL-6R) in both mice and rats, we find the highest expression of the IL-6R in the endocrine pancreas, with highest expression on the alpha-cell. The islet IL-6R is functional, and IL-6 acutely regulates both pro-glucagon mRNA and glucagon secretion in mouse and human islets, with no acute effect on insulin secretion. Furthermore, IL-6 stimulates alpha-cell proliferation, prevents apoptosis due to metabolic stress, and regulates alpha-cell mass in vivo. Using IL-6 KO mice fed a high-fat diet, we find that IL-6 is necessary for high-fat diet-induced increased alpha-cell mass, an effect that occurs early in response to diet change. Further, after high-fat diet feeding, IL-6 KO mice without expansion of alpha-cell mass display decreased fasting glucagon levels. However, despite these alpha-cell effects, high-fat feeding of IL-6 KO mice results in increased fed glycemia due to impaired insulin secretion, with unchanged insulin sensitivity and similar body weights. Thus, we conclude that IL-6 is necessary for the expansion of pancreatic alpha-cell mass in response to high-fat diet feeding, and we suggest that this expansion may be needed for functional beta-cell compensation to increased metabolic demand.

Induction of CXCL1 by extracellular matrix and autocrine enhancement by interleukin-1 in rat pancreatic beta-cells.

As we showed previously, the extracellular matrix (ECM) derived from rat bladder carcinoma cells (804G-ECM) has positive effects on rat primary beta-cell function and survival in vitro. The aim of this study was to define beta-cell genes induced by this ECM with a specific focus on cytokines. Analysis of differential gene expression by oligonucleotide microarrays, RT-PCR, and in situ hybridization was performed to identify cytokine mRNA induced by this matrix. Four cytokines were overexpressed on 804G-ECM compared with poly-L-lysine: C-X-C motif ligand 1 (CXCL1), CXCL2, interferon-inducible protein-10, and IL-1beta. A time-course experiment indicated that maximal induction by 804G-ECM of CXCL1/2 and interferon-inducible protein-10 occurred at 4 h. Stimulation of CXCL1 release by beta-cells on 804G-ECM was confirmed at the protein level. Moreover, secreted CXCL1 was shown to be functionally active by attracting rat granulocytes. Preventing the interaction of beta1 integrins and laminin-5 (a major component of 804G-ECM) with specific antibodies resulted in a 40-50% inhibition of CXCL1 expression. Using the nuclear factor-kappaB pathway inhibitor Bay 11-7082 it is demonstrated that CXCL1 expression and secretion are dependent on nuclear factor-kappaB activation. IL-1 secreted by beta-cells plated on 804G-ECM was found to be a key soluble mediator because treatment of cells with the IL-1 receptor antagonist significantly reduced both CXCL1 gene expression and secretion. It is concluded that ECM induces expression of cytokines including CXCL1 with amplification by IL-1 acting via a positive autocrine feedback loop.

Blockade of beta1 integrin-laminin-5 interaction affects spreading and insulin secretion of rat beta-cells attached on extracellular matrix.

When attached on a matrix produced by a rat bladder carcinoma cell line (804G matrix), rat pancreatic beta-cells spread in response to glucose and secrete more insulin compared with cells attached on poly-l-lysine. The aim of this study was to determine whether laminin-5 and its corresponding cell receptor beta1 integrin are implicated in these phenomena. By using specific blocking antibodies, we demonstrated that laminin-5 is the component present in 804G matrix responsible for the effect of 804G matrix on beta-cell function and spreading. When expression of two well-known laminin-5 ligands, beta1 and beta4 integrin, was assessed by Western blot and RT-PCR, only the beta1 integrin was detected in beta-cells. Anti-beta1 integrin antibody reduced the spreading of beta-cells on 804G matrix. Blockade of the interaction between beta1 integrins and laminin-5 resulted in a reduction in glucose-stimulated insulin secretion. Blocking anti-beta1 integrin antibody also inhibited focal adhesion kinase phosphorylation induced by 804G matrix. In conclusion, anti-beta1 integrin and -laminin-5 antibodies interfere with spreading of beta-cells, resulting in decreased insulin secretion in response to glucose. Our findings indicate that outside-in signaling via engagement of beta1 integrins by laminin-5 is an important component of normal beta-cell function.

Activation of NF-kappaB by extracellular matrix is involved in spreading and glucose-stimulated insulin secretion of pancreatic beta cells.

Laminin-5-rich extracellular matrix derived from 804G cells (804G-ECM) engages beta1 integrins to induce spreading, improve glucose-stimulated insulin secretion (GSIS), and increase survival of pancreatic beta cells. The present study examines whether 804G-ECM activates the transcriptional activity of NF-kappaB and the involvement of NF-kappaB in those effects of 804G-ECM on pancreatic beta cells. 804G-ECM induces nuclear translocation and the DNA binding activity of the p65 subunit of NF-kappaB. 804G-ECM-induced nuclear translocation of NF-kappaB was weak as compared with that induced by interleukin-1beta. Transient 804G-ECM-induced DNA binding activity of NF-kappaB (peak at 2 h) and overexpression of NF-kappaB target genes IkappaB alpha and NF-kappaB1(p105) (peak at 4 h) were observed. When NF-kappaB was inhibited by an inhibitor of IkappaB alpha phosphorylation (Bay 11-7082) or by a recombinant adenovirus expressing the nonphosphorylatable form of IkappaB alpha, 804G-ECM-induced cell spreading and actin cytoskeleton organization were reduced. GSIS from cells on 804G-ECM was inhibited 5-fold, whereas cell survival was not affected. In summary, the results indicate that 804G-ECM induces a transient and moderate NF-kappaB activity. This study shows for the first time that ECM-induced NF-kappaB activity is necessary in maintaining GSIS, although it does not affect survival of pancreatic beta cells. The effects of ECM-induced NF-kappaB activity contrast with the deleterious effects of cytokine-induced NF-kappaB activity. It is proposed that transient and moderate NF-kappaB activity is essential for proper function of the pancreatic beta cell.

Inhibition of calpain blocks pancreatic beta-cell spreading and insulin secretion.

In addition to promoting insulin secretion, an increase in cytosolic Ca(2+) triggered by glucose has been shown to be crucial for spreading of beta-cells attached on extracellular matrix (804G matrix). Calpains are Ca(2+)-dependent cysteine proteases involved in an extended spectrum of cellular responses, including cytoskeletal rearrangements and vesicular trafficking. The present work aimed to assess whether calpain is also implicated in the process of Ca(2+)-induced insulin secretion and spreading of rat pancreatic beta-cells. The results indicate calpain dependency of beta-cell spreading on 804G matrix. Indeed, treatment with three distinct calpain inhibitors (N-Ac-Leu-Leu-norleucinal, calpeptin, and ethyl(+)-(2S,3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl)butyl-carbamoyl]-2-ox-iranecarboxylate) inhibited cell spreading induced by glucose and KCl, whereas cell attachment was not significantly modified. Calpain inhibitors also suppressed glucose- and KCl-stimulated insulin secretion without affecting insulin synthesis. Washing the inhibitor out of the cell culture restored spreading on 804G matrix and insulin secretory response after 24 h. In addition, incubation with calpeptin did not affect insulin secretory response to mastoparan that acts on exocytosis downstream of intracellular calcium [Ca(2+)]i. Finally, calpeptin was shown to affect the [Ca(2+)]i response to glucose but not to KCl. In summary, the results show that inhibition of calpain blocks spreading and insulin secretion of primary pancreatic beta-cells. It is therefore suggested that calpain could be a mediator of Ca(2+)-induced-insulin secretion and beta-cell spreading.

Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression.

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (-/-) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/- and -/-) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/- and -/- embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4alpha, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.

Extracellular matrix protects pancreatic beta-cells against apoptosis: role of short- and long-term signaling pathways.

We have shown previously that culture of beta-cells on matrix derived from 804G cells and rich in laminin-5 improves their function. The purpose of this study was to investigate whether this matrix protects beta-cells against apoptosis and to elucidate signaling pathways involved. Matrix protected sorted rat beta-cells against apoptosis under standard conditions (11.2 mmol/l glucose, 10% serum), after serum deprivation (1% serum), and in response to interleukin-1beta (IL-1beta; 2 ng/ml), compared with control (poly-L-lysine [pLL]). Caspase-8 activity was reduced in cells cultured on matrix, whereas focal adhesion kinase (FAK), protein kinase B (PKB, or Akt), and extracellular signal-regulated kinase (ERK) phosphorylation was augmented. Treatment (4 h) with an anti-beta1 integrin antibody, with the ERK pathway inhibitor PD98059, and/or with the phosphatidylinositol 3-kinase inhibitor LY294002 augmented cell death on 804G matrix but not on pLL. In long-term assays (48 h), PD98059 but not LY294002 drastically augmented cell death on 804G matrix but did so to a lesser extent on pLL. The protein inhibitor of nuclear factor-kappaB (IkappaBalpha) was overexpressed in cells cultured 18 h on matrix with partial blockade by PD98059. In summary, this study provides evidence for activation of signaling pathways and gene expression by extracellular matrix leading to improved beta-cell survival.